The invention relates to a continuous closed system for the multi-step reaction, modification, production, and purification of proteins as well as synthetic analogs thereof.
A major task in chemical and biochemical syntheses concerns the purification of the desired reaction products from unwanted materials, including reagents, contaminants, by-products, solvents, etc. In a complex, multi-step synthesis, it is often necessary to conduct purifications at each of several critical steps in the synthetic process. Thus, separate isolation and purification procedures are usually employed for the reactant components, the intermediate and the final products of the synthesis with corresponding significant losses of material.
Such purifications must often be performed using specialized equipment and processes, which necessitates the transfer of reaction mixtures between reactant vessels and purification equipment. This is disadvantageous, because the methodology is inefficient and thereby complicates and prolongs manufacture. Moreover, the separate multi-step processes expose the product to the possibility of microbial and chemical contamination as well as the risk of degradation of the reagent or product at the various stages of synthesis thereby necessitating the inclusion of additional stringent process controls. This requirement for stringent process controls is particularly critical for the synthesis of pharmaceuticals.
As a consequence of the drawbacks of the prior art systems, there has been a pressing need for equipment which allows for the combination of the reaction and purification steps to the greatest degree possible. For example, peptide synthesis by solid phase methodology enables the intermediate purification steps to be conducted in the reaction vessel itself thereby reducing the presence of contaminant by-products. However, the complete purification of the cleaved peptide product may require several separate purification steps which result in loss of final product yield. (Stewart, et al. 1984 Solid Phase Peptide Synthesis; 2nd ed. Pierce Chemical Co., Rockford, Ill.)
The present invention provides a novel method that enables a general approach to liquid phase synthesis and product purification that is carried out in a single closed system.
The present invention is directed to a closed system for the continuous modification or conjugation of a protein and purification of the modified or conjugated protein product in a liquid phase. The system includes ultrafiltration apparatus, a reaction vessel, means for allowing the flow of the reaction solution from the reaction vessel to the ultrafiltration apparatus including the reverse flow from the ultrafiltration means and the reaction vessel, and a flow controlling means for regulating the flow thereof.
More than one ultrafiltration means may be incorporated for separating and purifying proteins and other molecules on the basis of molecular size in incremental orders of magnitude.
The closed system may also include a backwash reservoir which is fluidly connected to the ultrafiltration device for backwashing retained non-permeable peptides or proteins. The means for allowing the flow of reaction solution can be interconnecting tubing and the flow controlling unit may be made up of a pump and at least one valve. The pump in this system is preferably a circular or peristaltic type pump. The reaction vessel may also serve as a buffer reservoir in addition to a desalting reservoir.
The ultrafiltration means can be in the form of an ultrafiltration apparatus equipped with a spiral diafiltration cartridge, having a semi-permeable membrane having for example a molecular weight cutoff of 3,000; 5,000; 10,000; 15,000; 30,000 dalton; or higher. The aqueous system of the invention may also include a chromatographic device for separation of proteins from impurities or by-products by substrate affinity or size exclusion which is fluidly interconnected with reaction vessel and ultrafiltration device.
In certain embodiments the system of the invention may be automated with the use of electronical control means or may be entirely automated through the use of computerized process controls.
The invention provides a method for purifying a protein or a reaction product (coupled or conjugated protein) in a liquid closed system which comprises the steps of passing a protein or reaction mixture for the modification thereof in a liquid over a semipermeable membrane having a certain molecular retention cutoff contained in an ultrafiltration apparatus. The membrane selectively retains the protein or reaction product thereof on the basis of size. The protein or reaction product thereof retained on the membrane is washed extensively thereby removing the impurities which are not retained. The protein or peptide is then redispersed by backwashing or backflushing the membrane with the washing liquid, and concentrating and collecting the peptide or protein which is dissolved or suspended in the washing liquid. The concentrating, washing and backwashing cycles may be repeated for as many times as necessary for effective purification.